Biomimetic synergistic effect of redox site and Lewis acid for construction of efficient artificial enzyme

In enzymatic catalysis, the redox site and Lewis acid are the two main roles played by metal to assist amino acids. However, the reported enzyme mimics only focus on the redox-active metal as redox site, while the redox-inert metal as Lewis acid has, to the best of our knowledge, not been studied, presenting a bottleneck of enzyme mimics construction. Based on this, a series of highly efficient MxV2O5·nH2O peroxidase mimics with vanadium as redox site and alkaline-earth metal ion (M2+) as Lewis acid are reported. Experimental results and theoretical calculations indicate the peroxidase-mimicking activity of MxV2O5·nH2O show a periodic change with the Lewis acidity (ion potential) of M2+, revealing the mechanism of redox-inert M2+ regulating electron transfer of V-O through non-covalent polarization and thus promoting H2O2 adsorbate dissociation. The biomimetic synergetic effect of redox site and Lewis acid is expected to provide an inspiration for design of enzyme mimics.

Reviewer #2: Remarks to the Author: Lewis acids such as Ca2+ play multifaceted roles in biological enzymes, especially in the catalytic action of peroxidases, including stabilizing the active site structure, promoting substrate binding, regulating the catalytic process, stabilizing the enzyme-substrate complex, and adjusting enzyme activity.This provides a good perspective for the rational design of nanozymes, but current research on nanozyme designs that consider the auxiliary role of Lewis acids is limited, so the creativity of this paper is innovative.However, there are still some issues that need improvement, and I suggest considering publication in Nature Communications after major revision.Here are my suggestions: 1. Please clarify the long-term stability of the material under acidic conditions.Does the material degrade? 2. The theoretical calculation part of the mechanism does not consider the substrate, such as TMB.The adsorbed two HO* are directly desorbed, and one combines with H+ in the solution to get an electron from the material to form water and desorb.I don't think this mechanism is very reasonable because: a.The relative activity of the nanozyme is related to the substrate, so the substrate should be considered.b.The calculation results of this paper show that the adsorption of HO* is an endothermic process, so the free •OH adsorption on the surface is a spontaneous exothermic process.Therefore, compared to the mechanism of releasing free radicals, I think the process of continuously providing reductive hydrogen [H] to the two adsorbed HO* by reductive substrates, such as TMB, reported in the previous literature (Advanced Materials 2023, 2211151.) is more reasonable.3. The adsorption energy of H2O2 for noble metals, metal oxides, carbon-based materials, etc., is roughly between 0.0 and -1.0.However, the calculated adsorption energy of H2O2 in this paper is very strong, reaching -4.0 to -5.0 eV, which has obviously exceeded the strength of chemical adsorption, indicating that new chemical bonds have been formed.Please confirm the accuracy of the calculation, explain the reasons, or are there any literature reports of similar results? 4.There are already many theoretical reports on the mechanism of POD activity of nanozymes, but the authors rarely cite and discuss them. 5. Why didn't the authors consider the effect of surface Ca2+, instead of interlayer Ca2+, on enzyme activity?6.Using Lewis acid assistance to improve enzyme activity and perform structure-activity relationship analysis is an interesting topic, but the authors did not clearly explain the actual impact of Lewis acid on VO materials, simply explaining it as the polarization effect of ionic potential on VO.The effect from electronic effects is reasonable, but it should be thoroughly explained.I suggest that the authors calculate the band structure of the models and pay attention to the frontier energy levels, and discuss the activity in combination with the redox potential, because the essence of the substrate [H] snatching is a redox reaction.Therefore, the impact of Lewis acid on activity can be discussed from the perspective of the influence of electronic effects on the redox potential, which can be done by comparing the differential charge density analysis before and after addition, rather than simply analyzing the electron transfer of OH*, because there is no significant difference in the results of figure4Fc, d, e as it stands.7. Line 324-325, the authors did not calculate the transition state, so I don't understand the term "barrier of decomposition", and why the barriers are all negative, which seems inconsistent with the results in the figure.8. How did the authors calculate the charged system, by adding a background charge?Please supplement the explanation in the calculation method.
Reviewer #3: Remarks to the Author: This manuscript reported a series of MxV2O5•nH2O peroxidase mimics with vanadium as the redox site and redox-inert alkaline-earth metal ion (M2+) as the Lewis acid, which have more significant peroxidase-mimicking activity compared with other typical peroxidase mimics according to the maximal reaction velocity (Vmax) the turnover number (TON).The mechanism has been analyzed carefully by experiments and theoretical calculations.On this basis, MxV2O5•nH2O was used for antibacterial treatment and achieved remarkable effect.But some shortcomings also should be further corrected in the manuscript.I prefer to accept it after major revision.Some comments are listed as follows: 1.The quality of Fig 1C and D should be improved.Spherical aberration electron microscope is recommended.2. The biological safety of materials is very important, and it is suggested that the authors examine the safety of materials on normal cells, such as skin cells, vascular endothelial cells, fibroblasts, and so on.These experiments must be rigorous and substantial.3. I can not identify MxV2O5•nH2O nanomaterials in SEM images in Fig. S18.And the quality of SEM images of hydrogels were poor.4. The bacteria in the wound sites of different treatment groups need to be further characterized.5. What is the mechanism of material sterilization?Can hydroxyl radicals cross bacterial cell walls?Bacteria are more resistant to free radicals than mammalian cells due to the protection of cell wall.If the free radicals produced by the material are able to kill the bacteria, could the damage to the surrounding normal skin cells be greater?How to avoid damage to the normal cells of the wound?In addition, free radicals can cause wound inflammation.How to avoid the wound inflammation caused by free radicals?

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The authors of this manuscript have developed a series of alkaline earth metal (Ca, Mg, and Sr) intercalated V2O5 nanobelts as peroxidase mimics.This study was inspired by the role of alkaline earth metals present in the peroxidase enzyme, assisting in its activity.They have demonstrated that intercalation-induced geometric and electronic alterations affect vanadium centers, leading to the generation of high concentrations of oxygen vacancies.This effect enhances H2O2 activation to OH radicals, thereby exhibiting antibacterial activity.Consequently, this activity aids in wound healing in mice.The authors demonstrate the peroxidase-mimicking activity of MxV2O5•nH2O, showing a periodic change with the Lewis acidity (ion potential) of Ca, Mg, and Sr and this work has some potential.However, authors should consider other reports in the literature that have demonstrated the ability of alkaline earth metals, in combination with transition metals, to mimic peroxidase activity (see references: https://doi.org/10.1016/j.snb.2022.131869,https://doi.org/10.1016/j.mattod.2023.06.015, https://doi.org/10.1016/j.mtchem.2023.101861,https://doi.org/10.3390/chemosensors9080219).Although the current manuscript also deals with the antibacterial and wound-healing activities of their nanozymes, there exist numerous articles on this topic.This manuscript introduces some novel aspects, such as enhancing the activities of V2O5 nanobelts by non-redox metal intercalation.However, there are several issues to be addressed for improving this manuscript, as discussed below: Response: Thank you very much for your suggestions.With respect to "However, authors should consider other reports in the literature that have demonstrated the ability of alkaline earth metals, in combination with transition metals, to mimic peroxidase activity", alkaline earth metal ions as common cations may appear in many materials for charge balance or as templates/substrates.But so far, most of the enzyme mimics have focused on redox-active transition metals, and the role of redox-inert alkaline earth metals in enzyme-like catalysis have not been received much attention.The synergistic effect of transition metals and alkaline earth metals is also not proposed.We have carefully read and analyzed above references.In these papers, the simulated enzymes are SrMnO3, SrRuO3, SrFeO3 (Sens. Actuators, B, 2022, 364, 131869), calcium hexacyanoferrate (Ⅲ) nanoparticles (Mater. Today, 2023, 68, 148), FeCaP nanoparticles (Mater. Today Chem., 2024, 35, 101861) and Mg-Aminoclay-Based Fe3O4/TiO2 Hybrids (Chemosensors, 2021, 9, 219).The focus of these literatures lies in the application of Fe/Mn/Ru-based enzyme mimics, and there is no detailed exploration of structure-activity relationships.Importantly, alkaline earth metals exist in these materials just for charge balance (SrMnO3, SrRuO3, SrFeO3) and as substrates (calcium hexacyanoferrate (Ⅲ) nanoparticles, FeCaP nanoparticles and Mg-Aminoclay-Based Fe3O4/TiO2 Hybrids), and there is no further experimental validation or discussion about the role of alkaline earth metals in enzyme-like catalysis in these literatures.
We sincerely appreciate your recognition and comments on our work.The point-topoint responses to your comments are as follows: 1. What is the origin of the peak observed near 2θ = 10° in the XRD pattern?Response: The diffraction peaks near 2θ = 10° are attributed to the (001) crystal plane.The sharp diffraction peak of MxV2O5•nH2O near 2θ = 10° indicates that the crystal displays lamellar ordering as dominated by the pronounced (001) reflections (Chem, 2019(Chem, , 5, 1194)).With the intercalation of M 2+ and H2O in MxV2O5•nH2O, the lattice spacing of (001) increases, causing the diffraction peak of (001) shift to a smaller angle (Angew.Chem. Int. Ed., 2018, 57, 3943).By introducing the angle of diffraction peak into the Bragg equation 2dsinθ = nλ, the lattice spacing of (001) in MxV2O5•nH2O can be obtained.We have added the relevant description in the revised manuscript.
The manuscript was modified as follows: Page 6: In particular, compared with V2O5 powder and V2O5 nanobelt, sharp diffraction peaks before 2θ = 10° appeared in MxV2O5•nH2O (Fig. 1F, Supplementary Fig. 6).The diffraction peaks near 2θ = 10° are generally attributed to the (001) crystal plane, which indicate that the crystal displays lamellar ordering as dominated by the pronounced (001) reflections 40 .With the intercalation of M 2+ and H2O in MxV2O5•nH2O, the lattice spacing of (001) increases, causing the diffraction peak of (001) shift to a smaller angle 41 .
2. Although the authors have provided the length and width of the nanobelts, the thickness remains unspecified, which is crucial for defining them as nanobelts.It is recommended that the authors employ appropriate techniques such as AFM to characterize the structures thoroughly.Response: Thank you very much for your reminding.Although we preliminarily judged from SEM images that MxV2O5•nH2O have typical belt-like structure, we did not perform a rigorous characterization of them.In the revised manuscript, we added the AFM characterization of MxV2O5•nH2O (Supplementary Fig. 4).The results show that the thicknesses of most MxV2O5•nH2O materials are in the range of 20-60 nm, and significantly less than the widths of these materials.Due to their large aspect ratio and nanoscale thickness, the MxV2O5•nH2O materials are reasonably defined as MxV2O5•nH2O nanobelts.
The manuscript was modified as follows: Page 4: In addition, atomic force microscope (AFM) images show that the thicknesses of most MxV2O5•nH2O materials are in the range of 20-60 nm (Supplementary Fig. 4).Due to their large aspect ratio and nanoscale thickness, the MxV2O5•nH2O materials are also defined as MxV2O5•nH2O nanobelts.
The data were added in supporting information: 3. Despite the authors' indication of a width ranging from 200-300 nm based on SEM images, the TEM image distinctly reveals a width exceeding 500 nm.It is advised to review the TEM and EDX mapping images.Response: We are very sorry that the previous data are inaccurate.We used SEM to remeasure the width of MxV2O5•nH2O, and added the statistical results in Supplementary Fig. 3.According to the statistical results, the widths of MxV2O5•nH2O are mainly distributed in the range of 200 ~ 450 nm.The corresponding data have been changed in the revised manuscript.In addition, the TEM and EDX mapping images of MxV2O5•nH2O were replaced with more representative ones (Fig. 1B, E).In order to provide a more comprehensive view of the morphology and size of these materials, we also added TEM with a large field of view (Supplementary Fig. 2) in the supporting information.The manuscript was modified as follows: Page 4: The V2O5 nanobelt exhibits unique 1D structure with length of 1 to 5 μm and width of 10 to 100 nm (Supplementary Fig. 1B, D).The lengths of MxV2O5•nH2O are between 1 to 10 μm and the widths are between 200 to 450 nm (Fig. 1A, B, Supplementary Fig. 2, 3).The data were added in supporting information: Supplementary Fig. 2 The TEM images with a large field of view of MgVO (A), CaVO (B) and SrVO (C).Supplementary Fig. 3 The width distributions of MgVO (A), CaVO (B) and SrVO (C).
4. The key challenges faced by nanozymes are selectivity and specificity.These aspects are currently under active investigation by researchers working in the artificial enzyme area.While the authors assert that their nanozymes exhibit excellent and specific peroxidase-mimicking activity compared to oxidase, catalase, and SOD, previous studies suggest that various vanadium-based systems like nanomaterials and metalorganic frameworks also demonstrate haloperoxidase and glutathione peroxidase activities.Furthermore, there are reports highlighting the efficacy of highly efficient peroxidase nanozymes (https://www.nature.com/articles/s41467-022-32411-z).Response: According to your suggestion, we detected the haloperoxidase-mimicking activity and glutathione peroxidase-mimicking activity of MxV2O5•nH2O.As shown in Supplementary Fig. 15 and 16, MxV2O5•nH2O showed glutathione--mimicking peroxidase activity, and no significant haloperoxidase-mimicking activity was detected.
The manuscript was modified as follows: Page 9: In addition, previous studies have shown that some vanadium-based nanozymes also demonstrate haloperoxidase and glutathione peroxidase activities 49,50 .Therefore, we detected the haloperoxidase-mimicking activity and glutathione peroxidase-mimicking activity of MxV2O5•nH2O.As shown in Supplementary Fig. 15  5.While most nanozymes exhibit peroxidase-like activity through oxidation by OH radicals, this differs from natural peroxidase enzymes, which utilize ferryl oxo species for oxidation instead of OH radicals.Response: We are very sorry that we did not make a necessary discussion on the mechanism of POD-like nanozyme in the original text.We have added the mechanism discussion and related references in the revised manuscript.The definition of nanomaterials with POD-like activity usually means that the nanozymes are able to convert H2O2 into reactive oxygen species (ROS), which then oxidizes the substrate.Theoretical calculations and some experimental studies have showed that the catalytic reaction paths of POD-like nanozymes can be classified as two types according to the dissociation mode of H2O2 adsorbate (H2O2*) (Adv. Mater., 2024, 36, 2211151;ACS Nano 2024, 18, 19, 12367).The path 1 is that the dissociated H2O2* generates hydroxyl adsorbate (OH*) and hydroxyl radical (•OH), then generates H2O and oxidized substrate to complete the cycle (Nat. Nanotechnol., 2007, 2, 577;Angew. Chem. Int. Ed, 2019, 131, 4965;ACS Catal., 2020, 10, 6422;J. Am. Chem. Soc., 2021, 143, 2660;ACS nano, 2022, 16, 4536-4550).Because it is similar to the well-known Fenton reaction, this mechanism is also known as the Fenton-like mechanism and is widely accepted to explain the POD-like activity of many materials.In contrast, path 2 usually does not involve the formation of •OH.H2O2* first generates two OH*, which can directly oxidize the substrate under acidic conditions (ACS Catal., 2020, 10, 12657;J. Am. Chem. Soc., 2021, 143, 8855).Alternatively, OH* can also be converted to O* and H2O* through a hydrogen transfer reaction, with O* oxidizing the substrate (Biomaterials, 2015, 48, 37;J. Am. Chem. Soc., 2021, 143, 2660;Nat. Commun., 2022, 13, 4744).This path that adsorbed ROS directly acting on substrates has attracted more and more attention because it is similar to the catalytic mechanism of natural peroxidase (ferryl oxo species).In the revised manuscript, both mechanisms have been described, and corresponding theoretical calculations have also been supplemented and discussed.
The manuscript was modified as follows: Theoretical calculations and some experimental studies have showed that the catalytic reaction paths of POD-like nanozymes can be classified as two types according to dissociation mode of H2O2 adsorbate (H2O2*) 55,56 .The path 1 is that the dissociated H2O2* generates hydroxyl adsorbate (OH*) and hydroxyl radical (•OH), then generates H2O and oxidized substrate to complete the cycle 31,52,57-59 .Because it is similar to the well-known Fenton reaction, this mechanism is also known as the Fenton-like mechanism and is widely accepted to explain the POD-like activity of many materials.The path 2 usually does not involve the formation of •OH.H2O2* first generates two OH*, which can directly oxidize the substrate under acidic conditions 60,61 .Alternatively, OH* can also be converted to O* and H2O* through a hydrogen transfer reaction, with O* oxidizing the substrate 54,58,62,63 .This path that the adsorbed ROS directly acting on substrates has attracted more and more attention because it is similar to the catalytic mechanism of natural peroxidase (ferryl oxo species).In view of the direct detection of •OH produced under catalysis by ESR spectroscopy and the scavenging verification •OH by isopropyl alcohol, the possible catalytic mechanism of MxV2O5•nH2O is proposed following path 1.Therefore, the reaction mechanism was studied from the free energy, charge density difference, and band structure through density functional theory calculation.Subsequently, combined with the changes in the structure and catalytic activity of MxV2O5•nH2O after M 2+ intercalation, the synergistic effect of redox-inert M 2+ with redox-active V is further discussed.
Page 12: Besides the above mechanism involving releasing •OH, there are many reports on mechanism that the OH* oxidizes substrates directly under acidic conditions 60, 61 .In this path, H2O2* first generates two OH*, and then the reductive substrates (such as TMB) continuously providing reductive hydrogen [H] to the two adsorbed OH*.Since the presence of the substrate, it is considered to be more similar in energy to the actual catalytic process.Based on this, we calculate the free energy profile under this path.As shown in Supplementary Fig. 24, the dissociation of H2O2 is not affected after the substrate is added.Compared with V2O5 and V2O5•nH2O, the energy of H2O2 dissociation decreases significantly after Mg/Ca/Sr intercalation.This is consistent with the result in path 1 (Fig. 4B).The reaction of OH* with TMB-H + becomes completely spontaneous due to the formation of H2O.Therefore, from the perspective of free energy change, Ead (OH*), that is, the stability of OH* on the catalyst surface, is key to determine the catalytic reaction activity in both path 1 (Fig. 4B) and path 2 (Supplementary Fig. 24).
The data were added in supporting information: 6.The Vmax and KM value bars should be presented with error bars.Additionally, it should be noted that there are significant errors in evaluating the kinetic parameters.For instance, the Vmax for SrVO nanobelts is indicated as 71.7 x 10-8 M.s-1 (see supporting information Figure 9B(b)); however, this value is notably lower than the plateau region in the plot, which is not possible.Similarly, KM should ideally reflect the concentration at half of the actual Vmax, and these values significantly differ in most of the kinetic plots.Authors should carefully examine these aspects as these values are used for comparison with other reported nanozymes.Response: Thank you very much for your professional reminder.The Km and Vmax were calculated based on the mean values of the reaction rates by three independent steady-state kinetic assays and substrate concentrations.So, the error bars will not be presented in the calculated data.However, both the plots of reaction rates and the double reciprocal plot showed the errors bars caused by the three independent experiments, and detailed raw data about the kinetic parameter have been also provided in the Excel file named "Source Data".We have carefully checked the kinetic parameters in the original manuscript and found that some errors in the fitted data, especially for SrVO.In the revised version we have re-fitted all the dynamic parameters and modified the contents related to them.It should be emphasized that the re-fitted data show the relative order of Vmax corresponding to MxV2O5•nH2O (M = Mg, Ca, Sr) is same as that in the previous version, which is also consistent with the experimental results.
The manuscript was modified as follows: Page 9: As shown in Fig. 3F, when TMB was used as substrate, Vmax of MgVO, CaVO and SrVO nanobelts are 245, 220, 150 (10 -8 M•s -1 ), Km are 0.44, 0.54, 0.27 mM, respectively; When H2O2 was used as substrate (Fig. 3G), the Vmax of MgVO, CaVO and SrVO nanobelts are 306, 177, 148 (10 -8 M•s -1 ), and Km are 7.91, 6.46, 10.32 mM, respectively.Utilizing the quantity of materials while maintaining a constant surface area for all samples may be more relevant in this case.Response: When comparing the catalytic activity of nanozymes, the definition of nanozyme unit is crucial for different comparison methods.Among them, considering each atom/molecule in the nanoparticle as a nanozyme unit and each surface atom in the nanoparticle as a nanozyme unit are two common ways in the literatures (Adv. Mater., 2024, 36, 2211041).In general, considering each molecule in the nanoparticle (MxV2O5•nH2O, in this work) as a nanozyme unit is thought to result in underestimation of nanozyme activity.The method using surface atom as nanozyme unit (surface V, as your suggestion) is considered to better reflect the intrinsic catalytic activity of nanozymes under the premise that the activity depends only on the surface active sites.However, the determination of the surface atomic number first requires the surface area of the material, which can be obtained from the geometry calculations of nanomaterials or techniques such as Brunauer-Emmett Teller (BET) adsorption isotherms, and then the surface atomic number can be obtained according to the surface area and atomic arrangement of the crystal plane (ACS nano, 2021, 15, 15645).This way is usually suitable for nanoparticles with good monodispersity and uniform size.In this work, due to the large geometric heterogeneity of MxV2O5•nH2O, there may be significant errors in the measurement of surface atoms.In addition, it has been reported that the activity of nanozyme is not only determined by the surface atoms, the internal atoms may participate in catalytic regulation through electron transfer (Nat. Commun., 2022, 13, 5365).More importantly, in this work, since redox-inert M 2+ as Lewis acid participated in the POD-like catalysis of MxV2O5•nH2O, each MxV2O5•nH2O molecule in the particle as a nanozyme unit can better reflect the synergistic effect of M 2+ and V. Therefore, the activity comparison method adopted in this paper may be more suitable for our synthesized MxV2O5•nH2O.We have added relevant literature to the revised manuscript and accurately described the concentration of the nanozyme unit we used in the experimental method.
The manuscript was modified as follows: Page 11: In order to systematically evaluate its catalytic performance, we compared the Vmax and TON values (the maximum number of conversing substrates via the mole concentration of metal in the whole nanomaterials 51 ) of MxV2O5•nH2O with currently reported classical peroxidase mimics, Page 19: TON=Vmax/[E], where [E] is the molar concentration of MgVO/CaVO/SrVO.8.In Figure 4a, hydrogen peroxide should be adsorbed and not absorbed.This correction should be applied throughout the figure and text.Moreover, in step 2 of this figure, the term "hemlysis" used by the authors requires clarification.After step 3, the OH radical is depicted near the surface of the material, but its reaction with a proton (which should be proton adsorption) results in a water molecule.If this is the case, the generated water molecule would lack one electron, which is not feasible.Authors should carefully review the mechanism.Response: We are very sorry for the spelling errors.The "absorption" and "hemlysis" in Fig. 4 A and C have been revised to "adsorption" and "homolysis"，respectively.The corresponding words in the manuscript have also been checked and modified.For the actual catalysis, the electrons obtained by •OH generally come from the substrate or the catalyst (electron transfer).However, in the free energy profile in this paper, due to the use of calculation models that do not consider the substrate, the electrons directly derived from the substrate or the catalyst are not reflected in the mechanism diagram.Besides, in the free energy calculation using density functional theory, only the free energy of specific states such as H2O2 adsorption or OH adsorption was calculated, and no electron transport was involved.In addition, we have added the free energy profile considering TMB substrate (Supplementary Fig. 24), where the electrons are concerned As for the mechanism, we have added relevant content in the revised manuscript and introduced it in Response 5. Based on your suggestion and our further analysis on the reaction mechanism, the Fig. 4 A has been modified as followed: 10.In line 398, Authors say "the proposed MxV2O5•nH2O were extremely similar in structure and function to natural peroxidase".On what basis are they extremely similar in structure?The mechanism is also quite distinct from natural enzyme (please see comment no.5).Response: We are sorry for our imprecise description.In this paper, we emphasize that redox-active V and redox-inert M 2+ in MxV2O5•nH2O have good similarities with redox-active Fe 3+ and redox-inert Ca 2+ in natural horseradish peroxidase (HRP), in terms of structural and functional synergism of the two metals.For the structure, the HRP contains high-spin Fe 3+ , located in protoporphyrin IX in coordination with the proximal histidine ligand.At the same time, there is also redox-inert Ca 2+ in HRP-C.The Ca 2+ are on both sides of the protoporphyrin plane with a content of 2 mol of Ca 2+ /mol of enzyme.Although Fe 3+ and Ca 2+ are not chemically bonded, Ca 2+ can regulate the electronic structure of heme iron.In MxV2O5•nH2O, redox-inert M 2+ exist on both sides of the redox-active V as intercalation ions.At the same time, M 2+ and V are connected by non-covalent bond, but M 2+ can affect the electron density of V by ion polarization.So, the MxV2O5•nH2O is similar to HRP in geometric and electronic structure.For the catalytic function, the introduction of M 2+ effectively enhanced the POD-mimicking activity of MxV2O5•nH2O, partial removal of M 2+ leading to significant reduction of catalytic activity, which is also extremely similar to that, removal of Ca 2+ resulted in obvious decrease in the activity of the HRP-C (Biochem.Biophys.Res. Commun. 1978, 80, 1039;J. Biol. Chem. 1990, 265, 13335).The description of the structure had been presented in Scheme 1a.The function of M 2+ as Lewis acid had been experimentally validated.In order to make the expression more accurate, we have changed this sentence in the revised manuscript.
The manuscript was modified as follows: Page 14: According to the reported literatures, the redox-inert Ca 2+ as cofactors in natural peroxidase with Lewis acidity play a key role in the catalytic performance, which is mainly manifested in the loss of Ca 2+ leading to significant reduction of catalytic activity 16,17 .The structural similarities between MxV2O5•nH2O and natural peroxidase encouraged us to further investigate whether the redox-inert M 2+ could also act as cofactors like Ca 2+ of natural peroxidase to affect the peroxidase-mimicking activity of MxV2O5•nH2O.Thus, MxV2O5•nH2O were placed in heated alkaline liquid to remove the inserted M 2+ to achieve deintercalation.The results of ICP-MS showed that the molar ratios of Mg/V, Ca/V, and Sr/V in MxV2O5•nH2O decreased to 0.08/1, 0.13/1, and 0.14/1 respectively (Supplementary Fig. 26, Supplementary Table 4), confirming partial removal of M 2+ in MxV2O5•nH2O.The catalytic capacity of MxV2O5•nH2O for TMB oxidation before and after de-intercalation were then compared.From the result shown in Fig. 4G, the catalytic performance of MxV2O5•nH2O after deintercalation show obvious decrement owing to the decreased concentration of M 2+ .The SEM and XRD results show that the morphology and crystal structure of MxV2O5•nH2O after partial deintercalation have no obvious changes (Supplementary Fig. 27,28).The above results showed that the redox-inert M 2+ in MxV2O5•nH2O play similar roles to the Ca 2+ in natural peroxidase in regulating the catalytic reaction.Benefiting from these, the synergism of redox-active V and redox-inert M 2+ in MxV2O5•nH2O plays a key role in regulating the structure and function of peroxidase mimics, which is extremely similar to the relationship between redox site and Lewis acid in natural peroxidase.
11.The name of the bacteria should always be conventionally written in italics.For example, Staphylococcus aureus.Please correct such mistakes.Response: Thank you for your reminding.We have corrected such mistakes.
12. Authors have only used two bacteria in this study, but they claim that their nanozymes have broad-spectrum activity.Evaluation should be conducted using more strains to substantiate this claim.Response: Thank you for your advice.We added the antibacterial test for gram-positive Bacillus subtilis (B.subtilis) and gram-negative Pseudomonas aeruginosa (P.aeruginosa) by standard spread plate method.As shown in Supplementary Fig. 29,  13.I see that the nanozymes demonstrate antibacterial activities and not bacteriostatic activities.Kindly correct it in line 429.Response: Thank you very much for your reminding.We have made correction in the text and checked other similar words.The manuscript was modified as follows: Page 15: As shown in Fig. 5E, 5F, compared with other control groups, the red color of PI in the presence of both H2O2 and MxV2O5•nH2O was enhanced, indicating a significant antibacterial effect.
14.In Fig 6B, the dimensions of the wound (Day 0) in lanes 1, 2, and 3 appear to be different from the others.How can these be correctly compared in the study?Response: In infected wound healing experiments, although the wound creation followed same criteria, the errors were objectively present.We have shown the wound area statistics of multiple mice in different treatment groups in Supplementary Fig. 34, and the errors of the wound area are within a reasonable range.Because of the influence of shooting angle and mouse fixation, the dimensions of some wounds may look different in the photos.The antibacterial performance of the materials can be verified by the wound healing of the same mouse over a certain period of time.After applying the materials, all mice showed ideal wound healing.The manuscript was modified as follows: Page 15, 17: A round whole cortical wound with a diameter about 5.5 mm was formed on the back of each mouse (Supplementary Fig. 34) The data were added in supporting information: 15.In Supplementary Figure 18, no belt-like structures are visible in images B, C, and D. Considering that these belts have lengths of 1-4 μm, they are not visible at the scale size of 5 and 10 μm in the provided SEM images.Response: According to your suggestion, we re-tested the composite hydrogel by SEM.Since the GelTA is the main component of the composite hydrogel, MxV2O5•nH2O are only low content additives, it is difficult to directly observe the MxV2O5•nH2O in the composite hydrogel.In the revised support information, we replaced the previous SEM images with the SEM images containing EDX elemental quantification.The new data can show the elemental composition of the MxV2O5•nH2O functionalized composite hydrogel, as well as more typical porous structures.
The manuscript was modified as follows: Page 15: SEM images showed that freeze-dried GelTA has abundant porous structure with high interconnectivity.MxV2O5•nH2O functionalized GelTA (Mg-Gel-TA/ Ca-Gel-TA/ Sr-Gel-TA) also displayed similar porous structure, which were beneficial for exchange of substance (Supplementary Fig. 32).EDX elemental quantification shows the existence of MxV2O5•nH2O in the composite hydrogel.
The supporting information was modified as follows: Supplementary Fig. 32 The SEM images of GelTA (A) and SEM images with EDX elemental quantification (insets) of Mg-GelTA (B), Ca-GelTA (C), Sr-GelTA (D).
16. Authors have compared their nanozymes' activities with HRP and Fe3O4 reported by Gao et al.However, this comparison may not be appropriate as HRP and Fe3O4 have been evaluated under different reaction conditions.Therefore, it is improper to conclude that "the peroxidase-mimicking activity of MxV2O5•nH2O was more than one order of magnitude higher".
Response: We are very sorry for the inaccurate statement.The Vmax is only a reference index of enzyme activity and can not be used to directly compare the catalytical activity of materials.In the revised manuscript, we have revised the above sentence.The manuscript was modified as follows: Page 9-11: Comparing with Km and Vmax of the natural horseradish peroxidase (HRP) and classical Fe3O4 (Supplementary Table 2), the Km of MxV2O5•nH2O for the two substrates are similar to that of HRP and Fe3O4, but the Vmax of MxV2O5•nH2O are more than one order of magnitude higher than that of HRP and Fe3O4 under their respective optimal catalytic conditions.17.There are grammatical errors in multiple places.Response: Thank you very much for your reminding.We have re-checked the full text and made modifications to many non-standard sentences.
Reviewer #2 (Remarks to the Author): Lewis acids such as Ca 2+ play multifaceted roles in biological enzymes, especially in the catalytic action of peroxidases, including stabilizing the active site structure, promoting substrate binding, regulating the catalytic process, stabilizing the enzymesubstrate complex, and adjusting enzyme activity.This provides a good perspective for the rational design of nanozymes, but current research on nanozyme designs that consider the auxiliary role of Lewis acids is limited, so the creativity of this paper is innovative.However, there are still some issues that need improvement, and I suggest considering publication in Nature Communications after major revision.Here are my suggestions: 1. Please clarify the long-term stability of the material under acidic conditions.Does the material degrade?Response: Thanks very much for your reminding.In the revised manuscript, we have added the long-term stability verification of MxV2O5•nH2O at pH=5.The added XRD and SEM data (Supplementary Fig. 9, 10) showed that MxV2O5•nH2O maintained stable crystal structures and morphologies within 15 days.
The data were added in supporting information: to determine the catalytic reaction activity in both path 1 (Fig. 4B) and path 2 (Supplementary Fig. 24).At the same time, we still keep the free energy calculation without adding substrate according to the research goal of this work.In view of the complexity of the actual reaction, it is difficult for theoretical calculation to fully simulate the actual situation in the theoretical calculation.For POD-like nanozyme and natural POD, it has the ability to catalyze H2O2-dependent oxidation of a variety of substrates.Therefore, we believe that, under the condition without specific substrate, studying the intrinsic catalytic decomposition ability of nanozyme to H2O2 can also provide a valuable perspective for enzyme-like catalytic reaction mechanism.In this work, we focus on the biomimetic synergistic effect of redox site and Lewis acid, and try to propose a new strategy for constructing efficient artificial enzymes.The calculation ignoring substrate may have more advantages in analyzing material characteristics.In addition, we also revised the calculation models according to your comment 5 and 6, focusing on calculating the change of free energy before and after ion intercalation and discussing its effect on the catalytic activity of enzymes.
The manuscript was modified as follows: Page 12: Besides the above mechanism involving releasing •OH, there are many reports on mechanism that the OH* oxidizes substrates directly under acidic conditions 60,61 .In this path, H2O2* first generates two OH*, and then the reductive substrates (such as TMB) continuously providing reductive hydrogen [H] to the two adsorbed OH*.Since the presence of the substrate, it is considered to be more similar in energy to the actual catalytic process.Based on this, we calculate the free energy profile under this path.As shown in Supplementary Fig. 24, the dissociation of H2O2 is not affected after the substrate is added.Compared with V2O5 and V2O5•nH2O, the energy of H2O2 dissociation decreases significantly after Mg/Ca/Sr intercalation.This is consistent with the result in path 1 (Fig. 4B).The reaction of OH* with TMB-H + becomes completely spontaneous due to the formation of H2O.Therefore, from the perspective of free energy change, Ead (OH*), that is, the stability of OH* on the catalyst surface, is key to determine the catalytic reaction activity in both path 1 (Fig. 4B) and path 2 (Supplementary Fig. 24).
The data were added in supporting information: 3. The adsorption energy of H2O2 for noble metals, metal oxides, carbon-based materials, etc., is roughly between 0.0 and -1.0.However, the calculated adsorption energy of H2O2 in this paper is very strong, reaching -4.0 to -5.0 eV, which has obviously exceeded the strength of chemical adsorption, indicating that new chemical bonds have been formed.Please confirm the accuracy of the calculation, explain the reasons, or are there any literature reports of similar results?
Response: In the previous calculation, we built calculation models that included lattice oxygen vacancies.The simultaneous existence of lattice oxygen vacancy and surface unsaturated coordination may cause the adsorption models unstable.H2O2 will fill the oxygen vacancy after adsorption on the surface, resulting in low adsorption energy.In the revised draft, we remove the additional lattice oxygen vacancies to optimize the models.The adsorption energy of H2O2 for each model is within the reasonable range of 0.0 to -1.0.In addition, according to your comment 5 and 6, we modified the relevant crystal models and added a new control model to obtain a new free energy profile.The relevant content has been added in the main text.The manuscript was modified as follows: 4.There are already many theoretical reports on the mechanism of POD activity of nanozymes, but the authors rarely cite and discuss them.Response: Thank you very much for your suggestion, we have added relevant content and literature in the revised manuscript.The definition of nanomaterials with POD-like activity usually means that the nanozymes are able to convert H2O2 into reactive oxygen species (ROS), which then oxidizes the substrate.Theoretical calculations and some experimental studies have showed that the catalytic reaction paths of POD-like nanozymes can be classified as two types according to dissociation mode of H2O2 adsorbate (H2O2*) (Adv. Mater., 2024, 36, 211151;ACS Nano 2024, 18, 12367).The path 1 is that the dissociated H2O2* generates hydroxyl adsorbate (OH*) and hydroxyl radical (•OH), and generates H2O and oxidized substrate to complete the cycle (Nat. Nanotechnol., 2007, 2, 577;Angew. Chem. Int. Ed, 2019, 131, 4965;ACS Catal., 2020, 10, 6422;J. Am. Chem. Soc., 2021, 143, 2660;ACS nano, 2022, 16, 4536).Because it is similar to the well-known Fenton reaction, this mechanism is also known as the Fenton-like mechanism and is widely accepted to explain the POD-like activity of many materials.Path 2 usually does not involve the formation of •OH.H2O2* first generates two OH*, which can directly oxidize the substrate under acidic conditions (ACS Catal., 2020, 10, 12657;J. Am. Chem. Soc., 2021, 143, 8855).Alternatively, OH* can also be converted to O* and H2O* through a hydrogen transfer reaction, with O* oxidizing the substrate (Biomaterials, 2015, 48, 37;J. Am. Chem. Soc., 2021, 143, 2660;Nat. Commun., 2022, 13, 4744).This path that the adsorbed ROS directly acting on substrates has attracted more and more attention because it is similar to the catalytic mechanism of natural peroxidase (ferryl oxo species).
The manuscript was modified as follows: Page 11: Theoretical calculations and some experimental studies have showed that the catalytic reaction paths of POD-like nanozymes can be classified as two types according to dissociation mode of H2O2 adsorbate (H2O2*) 55,56 .The path 1 is that the dissociated H2O2* generates hydroxyl adsorbate (OH*) and hydroxyl radical (•OH), then generates H2O and oxidized substrate to complete the cycle 31,52,57-59 .Because it is similar to the well-known Fenton reaction, this mechanism is also known as the Fenton-like mechanism and is widely accepted to explain the POD-like activity of many materials.The path 2 usually does not involve the formation of •OH.H2O2* first generates two OH*, which can directly oxidize the substrate under acidic conditions 60,61 .Alternatively, OH* can also be converted to O* and H2O* through a hydrogen transfer reaction, with O* oxidizing the substrate 54,58,62,63 .This path that the adsorbed ROS directly acting on substrates has attracted more and more attention because it is similar to the catalytic mechanism of natural peroxidase (ferryl oxo species).In view of the direct detection of •OH produced under catalysis by ESR spectroscopy and the scavenging verification •OH by isopropyl alcohol, the possible catalytic mechanism of MxV2O5•nH2O is proposed following path 1.Therefore, the reaction mechanism was studied from the free energy, charge density difference, and band structure through density functional theory calculation.Subsequently, combined with the changes in the structure and catalytic activity of MxV2O5•nH2O after M 2+ intercalation, the synergistic effect of redox-inert M 2+ with redox-active V is further discussed.5. Why didn't the authors consider the effect of surface Ca 2+ , instead of interlayer Ca 2+ , on enzyme activity?Response: Due to the large difference in valence state and outer electronic structure between alkali earth metal M and transition metal V, M 2+ is theoretically not inclined to be incorporated into the host lattice of V2O5 in the form of covalent bonds.The M 2+ in MxV2O5•nH2O is an intercalating doping based on non-covalent bonding, which is verified by XRD and XPS.This weak interaction is relatively stable between layers, but unstable on the surface.In addition, M 2+ is considered to have no catalytic ability.The redox-inert M 2+ should improve the POD-like catalytic activity by regulating electronic structure of V. Therefore, interlayer M 2+ , as the main existing form of M 2+ in the layered materials, is usually not considered to directly contact substrates on material surface, but rather achieves synergistic effect with surface V. 6.Using Lewis acid assistance to improve enzyme activity and perform structure-activity relationship analysis is an interesting topic, but the authors did not clearly explain the actual impact of Lewis acid on VO materials, simply explaining it as the polarization effect of ionic potential on VO.The effect from electronic effects is reasonable, but it should be thoroughly explained.I suggest that the authors calculate the band structure of the models and pay attention to the frontier energy levels, and discuss the activity in combination with the redox potential, because the essence of the substrate [H] snatching is a redox reaction.Therefore, the impact of Lewis acid on activity can be discussed from the perspective of the influence of electronic effects on the redox potential, which can be done by comparing the differential charge density analysis before and after addition, rather than simply analyzing the electron transfer of OH*, because there is no significant difference in the results of figure 4Fc, d, e as it stands.
Response: According to your suggestion, we calculated the frontier molecular orbitals (FMO) of the materials before and after intercalation.Then, the changes of POD-like activity were analyzed by combining the band structures and the redox potential of POD-like catalytic reaction (Nat. Commun., 2021, 12, 6866;ACS Appl. Mater. Interfaces, 2014, 6, 1959).The experimental results showed that the FMO energy of the material moved towards negative potential after Lewis acid intercalation, indicating that the reduction ability of MxV2O5•nH2O was significantly enhanced.At the same time, the valence orbital maximums of MxV2O5•nH2O are close to or lower than the reduction potential of H2O2 (Fig. 5 F).According to relevant literature (Nat. Commun., 2021, 12, 6866;ACS Appl. Mater. Interfaces, 2014, 6, 1959) and Nernst equation, this band structure is conducive to electron transfer in the catalytic reaction to achieve enzyme-like catalysis.We also analyzed the intrinsic barder charge of the material before and after the Lewis acid intercalation.The experimental results show that the polarization of Lewis acid can reduce the electron density around V, make V more inclined to form V-OH, and promote the H2O2 homolysis (Supplementary Fig. 25).The manuscript was modified as follows Page 13: In order to further understand the effect of Lewis acid intercalation on enzymemimicking catalytic activity, the band structure before and after intercalation were calculated and discussed combing with the POD-like catalytic redox potential 66,67 .The typical reaction catalyzed by peroxidase nanozyme is as follows: H2O2+2TMB + 2H + = 2H2O +2TMB + , where TMB + is the oxidation state of TMB.The reaction can be divided into two half reactions as follows: TMB + + e -= TMB, φ1; 1/2H2O2 + H + + e -= H2O, φ2 The reduction potential of TMB + /TMB (φ1) used is about 1.13V, referring to a wellestablished value in the literature, and the standard reduction potential of H2O2/H2O (φ2) is 1.776V 68,69 .As can be seen from Fig. 4F, the frontier molecular orbitals (FMO) of V2O5 before intercalation, including the valence band maximum (VBM) and the conduction band minimum (CBM), display more positive energy than φ2, indicating that electrons can be transferred from TMB to V2O5, but cannot be transferred from V2O5 to H2O2 to complete the catalytic reaction.After Lewis acid M 2+ intercalation, the energies of the FMO of MxV2O5•nH2O move significantly toward the negative direction, indicating that the reduction abilities of MxV2O5•nH2O are significantly enhanced.Among them, the energies of VBM is close to or less negative than φ2, indicating that electrons can be passed from TMB to V2O5, and then to H2O2 to complete the catalysis.The H2O intercalated V2O5•nH2O shows a more negative FMO energy, and electrons cannot transfer from TMB to V2O5.Then, the influence of Lewis acid M 2+ on the intrinsic electronic structure of redoxactive V is further analyzed by calculating the average bader charge of V before and after intercalation.As shown in Supplementary Fig. 25, the bader charge around V in V2O5 before intercalation is 2.1670 |e|.After H2O intercalation, the bader charge around V is 2.1730 |e|, and no significant change occurs.After M 2+ intercalation, the average bader charge around V decreases to 2.1379, 2.1264 and 2.1242 |e|, respectively.Since M 2+ acts directly on the coordinated O of V through electrostatic attraction, the polarization of M 2+ on the V-O bond results in the decrease in electron density around V. In general, V with low electron density will be more inclined to produce stable V-OH*.Thus, the Lewis acid M 2+ promotes the H2O2* homolysis by adjusting the electronic structure of V.
The data were added in supporting information: About the damage to the normal cells: As with most ROS based antibacterial materials, ROS produced by the MxV2O5•nH2O will inevitably damage the cells close to the hydrogel on the wound surface, but the death and regeneration of these cells is a common phenomenon in the wound healing process.In the group treated by MxV2O5•nH2O functionalized GelTA, the wound healing was obvious (Fig. 6B), the epidermal layer (Fig. 6F) and collagen fibers (Fig. 6G) were intact, indicating that MxV2O5•nH2O in the hydrogel had no obvious killing effect on tissue cells.

About the wound inflammation:
The composite hydrogel exists as a wound dressing, essentially creating a barrier between the environment and the wound.MxV2O5•nH2O act as antibacterial additives to kill bacteria that try to enter the wound surface.Due to the stability of the hydrogel and the large size, MxV2O5•nH2O does not tend to enter tissues and cause systemic oxidative stress, which can be confirmed by Fig. 6F.There is no significant inflammatory reaction in the healed tissues of mice in the treatment groups.The above content has been discussed in the article.
Page 14, 15: The enzyme mimics are widely used in antibacterial researches, because the reactive oxygen species (ROS) catalyzed by enzyme mimics can oxidize key components of bacteria, such as cell membranes/walls or intracellular compartments 70-72 .
Page 17: Further, Hematoxylin and eosin (H&E) staining was analyzed for the wound tissue of different groups of mice after seven days of treatment (Fig. 6F).The results show that the epidermal layers at the wound sites are more intact in the treatment groups, and only a few inflammatory cells are distributed.In the control groups, the epidermal layers at the wound sites are fragmented and many inflammatory cells gather in the wound areas.
Masson staining shows that the density of collagen fibers at the wound site are higher in the treatment groups than in the control groups (Fig. 6G).
and 16, MxV2O5•nH2O show glutathione-mimicking peroxidase activity, and no significant haloperoxidasemimicking activity was detected.The data were added in supporting information: Supplementary Fig. 15 Exploration of glutathione peroxidase-mimicking activity of MxV2O5•nH2O.Timedependent UV-vis absorption spectra for monitoring the glutathione peroxidase-mimicking catalytic activities of MgVO (A), CaVO (B) and SrVO (C), under the condition of phosphate buffer (pH 7.4) containing 2 mM GSH, 0.4 mM H2O2, and 0.4 mM NADPH at room temperature.Time-dependent UV-vis absorption spectra for monitoring the glutathione peroxidase-mimicking catalytic activities of MxV2O5•nH2O, under the above condition without GSH (D) or without H2O2 (E).Supplementary Fig. 16 Exploration of haloperoxidase-mimicking activity of MxV2O5•nH2O.Time-dependent UVvis absorption spectra for monitoring the glutathione peroxidase-mimicking catalytic activities of MgVO (A), CaVO (B) and SrVO (C), under the condition of NaAc/HAc buffer (pH = 5.0) containing 28 μM phenol red, 4.4 mM NH4Br, and 0.42 mM H2O2 at room temperature.

Fig. 4
Fig. 4 (A) The possible reaction pathway on M x V 2 O 5 •nH 2 O, taking MgVO for example.(Purple: V atom; Red: O atom; White: H atom; Yellow: Mg atom).
Supplementary Fig. 34.The diameters of wounds at day 0 measured by vernier caliper.ns represents no statistical difference.Error bars represent ±SD (n = 3)

Fig. 4 .
Fig. 4. (B) Free energy diagram of the proposed reaction pathway for V2O5, V2O5•nH2O and MxV2O5•nH2O models.The data were added in supporting information:

Fig. 5
Fig. 5 (F) Calculated electronic density of states with the energies of FMOs marked